1. Field of the Invention
The invention generally relates to methods of producing ω-3 fatty acids using microalgae. In particular, the invention provides a multiphase method for high density heterotrophic microalgal growth and production of ω-3 fatty acids. The multiphase method decouples cell division, cell growth, and ω-3 fatty acid production by controlling optimal conditions for each in separate reactors. The decoupling and optimization result in overall improvements in algal, lipid, and polyunsaturated fatty acid (PUFA) yield and productivity. In addition, the process has been integrated to a biodiesel refinery by utilizing waste feedstocks such as crude glycerol, a byproduct of biofuel generation, as the primary carbon source. Use of in situ transesterification and fractional distillation processes allow for products from the algal growth process to include not only PUFA lipids generated by the microalgae, but also non-PUFA lipids which can be used in biofuel generation, thereby forming an integrated microalgae-biofuel system for sale of biodiesel methyl esters and PUFA in a distinguishing methyl ester form.
2. Background of the Invention
Recent sociopolitical and economic developments have highlighted the need for efficient and scalable methods for bioproduct generation and utilization. While much energy has focused on the utilization of existing agricultural products, such approaches have non-desirable secondary effects. Specifically, the diversion of productive cropland from food production to the production of useful chemicals and materials ultimately results in an increase in product costs. This has led to enhanced interest in the development of methods for the generation of valuable products from marginal land and/or from waste streams or low value materials (Gonzalez-Pajuelo, 2006; Papanikolaou, 2002; Narayan, 2005; Meesters, 1996). For example, crude glycerol (glycerol is also referred to as “glycerin”) is a byproduct of the transesterification reaction that is used to make biodiesel from plant sources. Due to recent increases in biofuel production, the world market is currently experiencing an enormous glut of crude glycerol. Various uses of crude glycerol have been proposed. Purification can be carried out to produce purified glycerol; crude glycerol can be used in animal feed (especially for pigs); and crude glycerol may be used as a substrate for fermentation by anaerobic bacteria (e.g. Clostridium sp.) to produce useful products such as lactic acid, propionic acid, etc., as well as active programs to convert waste glycerol to antifreeze, bioploymer precursors and soaps. However, excess crude glycerol is still being produced and is generally disposed of by simply burning it, a wasteful process. A need exists to develop additional methods for dealing constructively with “waste” crude glycerol.
Another example is the renewed interest in the harvesting of products derived from algae, particularly from heterotrophic microalgae with known capabilities for generating and storing within their biomass large quantities of lipids, and in particular omega-3 lipids with known health benefits. Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) are a group of fatty acids containing two or more double bonds, of which the last double bond is located at the third carbon atom from the methyl terminal. For a long time, the beneficial effects of ω-3 PUFAs have been recognized by epidemiological surveys that revealed that Eskimos, who consume large amounts of deep-sea fish, rarely suffered from heart diseases. Docosahexaenoic acid (DHA, 22:6), with a 22 carbon chain and 6 double bonds, is one of the more important (ω-3 PUFAs and is known to have particular beneficial effects in fetal and infant brain and ocular development. The inclusion of supplementary DHA in infant formulas is strongly recommended by the World Health Organization (WHO) (FAO/WHO Expert Committee, 1994). Also, research continues to demonstrate the need for DHA beyond infancy, with studies suggesting a positive correlation between DHA consumption and the reduced risk of age-related neurological disorders, such as Alzheimer's and dementia (Ward and Singh, 2005). As a result, DHA is not only used as an additive in infant formulas, but also in adult dietary food and beverages. Examples include cheeses, yogurts, spreads and dressings, and breakfast cereals. Notably, these markets may have much greater growth potential than infant formulae, thereby substantially elevating the entire DHA market potential (Ward and Singh, 2005).
The conventional source of ω-3 PUFAs is predominantly fish oil and seal oil. Cod, salmon, sardine, mackerel, menhaden, anchovy, and tuna are generally used for fish oil production. The quality of fish oil depends on the fish species, the season and the geographical location of catching sites. As marine fish oil is a complex mixture of fatty acids with varying chain lengths and degrees of unsaturation, DHA must be refined from fish oil for use in nutraceutical/pharmaceutical applications. The purification of DHA from low-grade fish oil is difficult and costly (Belarbi et al., 2000). In addition, marine fish stocks are subjected to seasonal and climatic variations, and may not meet the requirement for providing a steady supply for the increasing demands of DHA.
It is known that fish, like humans, are not capable of synthesizing PUFA de novo. Much of their PUFA is derived from the primary producer in the oceanic environment: microalgae or algae-like microorganisms. There are a large number of microalgae in nature which produce PUFAs in general and DHA in particular, but only a few species have demonstrated production potential on an industrial scale. This is mainly due to the low specific growth rates and low cell density of the algae, since in many cases they can only grow under photoautotrophic conditions.
Intensive research into the production capabilities of these microalgae led researchers and commercial industries to focus on and develop heterotrophic algal production processes for DHA. As of late, the two algae used commercially and showing the greatest commercial promise are the heterotrophic dinoflagellate Crythecodinium cohnii and strains of traustochytrid marine protists. Developments of commercial processes for production of DHA with these two algae has benefited from the fact that they can accumulate high oil contents in their biomass (10-50%, w/w) and produce a high percentage of total lipids as DHA (30-70%). High biomass densities (up to 109 g/L) and DHA concentrations of 20 g/L have been achieved in carbon fed batch cultures of the marine species, C. cohnii, although prolonged culture times (400 h) were required. Studies have demonstrated that DHA productivities of 1-1.5 g/(L day) are achievable with this strain (Ward and Singh, 2005).
However, the best microbial sources of DHA are Thraustochytrids, specifically the genera Thraustochytrium and Schizochytrium. Thraustochytrium and Schizochytrium are unicellular algal or algal-like protists, members of the order Thraustochytriales; family Thraustochytriaceae; genus Thraustochytrium or Schizochytrium. Schizochytrium replicates by both successive bipartition and by release of zoospores from sporangia, whereas Thraustochytrium strains only replicate by formation of sporangia/zoospores. Studies with thraustochytrids have established these marine protists as preeminent industrial strains for the production of DHA. Initial research at relatively low cell densities (5-20 g/L) established the capacities of Thraustochytrium species to accumulate greater than 50% of their lipids as DHA and to produce >1 g DHA/L of culture, with productivities of about 0.2 g/(Lday) (Ward and Singh, 2005). Schizochytrium species with even higher growth rates have been isolated. Under glucose and nitrogen-fed batch conditions, with incorporation of sodium sulfate as a main sodium source and with control of glucose concentrations, pH and oxygen levels, selected strains have been shown to grow to high biomass densities (100 g/L) in short fermentation cycles (90-100 h), accumulating 4045 g/L of DHA and DHA productivities of >10 g/(L day). These excellent performances have made Schizochytrium the producer of choice in the DHA industry (Bailey et al., 2003).
Specific industrial utilization and commercial culture conditions of the genera Thraustochytrium or Schizochytrium for the generation of PUFA products is discussed in detail in U.S. Pat. No. 5,130,242 (Barclay, which is incorporated herein by reference). In '242 and related filings, U.S. Pat. Nos. 5,340,742 and 6,977,167, (both to Barclay, the complete contents of both of which are hereby incorporated by reference) algae from these genera are cultured in closed reactors under controlled salt, oxygen, and temperature, and carbon and nitrogen source concentrations which were tailored to maximize the production of the PUFA product. However, the carbon and nitrogen sources were derived solely from relatively high-value agricultural products, i.e. the use of crude glycerol is not discussed.
Within these disclosures much focus has been directed toward the increase in biomass and PUFA product yield. In U.S. Pat. Nos. 5,130,242 and 6,977,167 it was disclosed that, through the maintenance of a relatively high concentration of phosphate, a sustained growth can be maintained, which enables, in part, the production of a high density culture. Moreover, through the specific control of the nitrogen source, or when the nitrogen source becomes limited (either through controlled addition or static initial supply) for some time period prior to cell harvesting the biomass will have an increased concentration of PUFA. Similarly, in U.S. Pat. No. 6,607,900 (Bailey et al., the complete contents of which are hereby incorporated by reference) a two phase fed-batch fermentation process, wherein a first phase aimed at biomass density is followed by a second lipid production phase, is disclosed. As described in '900, and similar to the process disclosed in U.S. Pat. Nos. 5,130,242 and 6,977,167, the biomass density phase comprises a fermentation medium containing sugar and amino acid derivatives (as a nitrogen source) and the lipid accumulation phase comprises mostly sugar with limited amino acid derivatives within the fermentation medium, with the added caveat that the oxygen concentration during the lipid accumulation phase is less than the oxygen concentration during the biomass accumulation phase.
While relatively little attention has been directed toward the specific functional relationships between the nutrient variables and the cell state, some information is available. In U.S. Pat. No. 5,340,742, growth methods are described wherein non-chloride salts, specifically sodium sulfate and/or low chloride concentrations, were utilized to provide 1) decreased corrosion of the fermentation and cell harvesting hardware and 2) a smaller aggregate cell size while maintaining a high PUFA concentration. Using the process described in U.S. Pat. No. 6,607,900, with the fed-batch mode, at least 100 g/L of biomass density can be obtained by feeding the carbon source and the nitrogen source at a sufficient rate. In this process, the fermentation condition comprises a biomass density increasing stage and a lipid production stage. Both the carbon source and the nitrogen source are added in the biomass density increasing stage, while only a carbon source is added during the lipid production stage. The level of dissolved oxygen in the medium during the fermentation biomass density increasing stage is at least about 4%, and during the lipid production stage is less than about 3%. The preferred temperature in this process is about 30° C. The lipid production rate of this process can reach 1.0 g/L/hr, and the DHA in the lipids is about 40%. On the whole, according to U.S. Pat. No. 6,607,900, the DHA production rate can reach 0.5 g/L/hr.
In summary, U.S. Pat. No. 6,607,900 teaches culturing microalgae (e.g. Thraustochtriales of the genera Thraustochytrium and Schizochytrium) in two stages. The first is a biomass density increasing stage during which the dissolved oxygen is at least about 8% and preferably about 4%. In contrast, in the second “production” stage, during which the primary activity of the algae is not increasing biomass but producing lipids, the amount of oxygen is decreased to about 1% or preferably 0%. U.S. Pat. No. 6,607,900 teaches using temperatures of at least 20° C., more preferably 25° C., and most preferably 30° C., without specifying which temperatures are suitable for which stage. However, U.S. Pat. No. 6,607,900 states that, because cold water can retain a higher amount of dissolved oxygen than warm water, a “higher fermentation medium temperature has the additional benefit of reducing the amount of dissolved oxygen . . . ”. Thus, U.S. Pat. No. 6,607,900 suggests that higher temperatures are advantageous during the production stage when low or no oxygen is preferred. U.S. Pat. No. 6,607,900 also teaches a total fermentation time of from about 90 to about 100 hours. In addition, U.S. Pat. No. 6,607,900 teaches that the carbon source for the process is preferably nonalcoholic, and preferably a carbohydrate such as corn syrup. These teachings would suggest ruling out the use of crude glycerol (glycerol is an alcohol).
In addition, U.S. Pat. No. 5,130,242 discusses a two-stage algae fermentation process to produce lipids. The first stage is an exponential growth stage, and the second is a stationary lipid production stage. During the latter, nitrogen limitation stimulates lipid production. However, U.S. Pat. No. 5,130,242 teaches that the nitrogen-limited period should be relatively short, e.g. 6-24 hours. According to U.S. Pat. No. 5,130,242, the preferred nutritive sources for both carbon and nitrogen are grains and certain hydrolyzed waste products such as stillage, a waste product in corn to alcohol fermentations. However, the use of crude glycerol is not discussed. “Crude glycerol” differs from “glycerol” in that, as a byproduct of the biodiesel production process, crude glyderol contains roughly 70-80% glycerol mixed with industrial process contaminants such as free fatty acid anions, mono and di-glycerides, alcohol and salts. These substances come from the raw material or from incomplete reactions during the fuel production process.
Further, U.S. Pat. No. 6,582,941 (Yokochi et al.) discloses Schizochytrium genus strain SR21 as capable of producing highly unsaturated fatty acids. Glycerol is one suggested carbon source. However, the use of byproducts such a crude glycerol is not discussed. (See also Yokochi et al., 1998.)
Finally, International patent application WO 2004/083442 (Kumar et al.) describes a method of increasing the levels of PUFAs in cultured thraustochytrids by storing them in the cold after 2-5 days of growth. Crude glycerol is not suggested as a carbon source.